This invention relates generally to microbicidal compounds and, more particularly, to broad spectrum tryptophan-rich peptide known as indolicidin.
Infectious diseases are a primary cause of morbidity and mortality in humans and animals. For example, 8 to 10 million people have been estimated to be infected with the AIDS virus with 263,000 new cases reported in 1990 alone. Many persons infected with the AIDS virus will further suffer from opportunistic infections, such as Candida albicans, which causes mucocutaneous fungal disease. Other microbial infections include, for example, E. coli diarrhea which is caused by consumption of contaminated food and drinks. This infection affects 40-50% of visitors from industrialized countries travelling to developing countries. Gonorrhea, which is caused by a gram negative bacterium, was reported in over seven hundred and fifteen thousand cases in the United States in 1990, and 3,000 to 10,000 new cases per 100,000 people are diagnosed per year in Africa.
Antibiotic resistant strains of E. coli as well as other bacterial, viral, and fungal pathogens, make treatment of many diseases difficult and expensive. Even in cases where a disease may potentially be treated by antibiotics, the unavailability of adequate storage facilities for antibiotics, especially in underdeveloped regions of the world where diseases often are endemic, results in the inability to provide effective treatment to infected populations.
In vertebrates, polymorphonuclear leukocytes, including neutrophils and granulocytes have a central role in combatting infectious disease. These white blood cells contain membrane-bound, cytoplasmic granules, which store various components of their microbicidal arsenal. Upon infection, neutrophils engulf the invading microorganisms in membrane-bound vesicles. These vesicles then fuse with the cytoplasmic granules, exposing the microbes to the toxic contents of the granules. One mechanism granulocytes have for killing such microbes consists of an array of peptides that act as naturally-occurring antibiotics. These peptides, which are generally cationic, mediate their toxicity by interacting with and permeabilizing the cell membranes of various microorganism.
Two families of microbicidal peptides have previously been isolated from granulocytes. The bactenecins, described by Genarro et al., Infect. Immun. 57:3142-46 (1989), Romeo et al., J. Biol. Chem. 263:9573-75 (1988), and Marzari et al, Infect. Immun. 56:2193-97 (1988), are proline and arginine-rich peptides that range in size from 1600 to 8000 daltons which were identified in part by their reactivity with a monoclonal antibody raised against a granule protein extract. The bactenecins are toxic to fungi and gram negative bacteria and, to a lesser extent, to gram positive bacteria.
The defensins are a family of fifteen peptides which constitute 5% to 18% of the cellular protein in neutrophils of various species. This class of molecules has been described by Ganz et al., Eur. J. Haematol. 44:1-8 (1990), Lehrer et al., U.S. Pat. Ser. No. 4,543,252, and Selsted et al., Infect. Immun. 45:150-154 (1984). The defensin peptides consist of 29 to 34 amino acids, with four to ten of these residues being arginine. In addition, they all share six conserved cysteine residues that participate in intramolecular disulfide bonds. Defensins are microbicidal to gram negative and gram positive bacteria, fungi, and certain enveloped viruses.
While the availability of naturally-occurring antibiotic peptides is extremely valuable for treating infectious diseases that are not otherwise amenable to treatment by synthetic antibiotics, the usefulness of bactenecins and defensins suffers from various limitations. For example, both bactenecins and defensins are immunogenic and, therefore, treatment using these compounds could potentially result in anaphylactic or delayed hypersensitivity-type responses. The defensins have also been demonstrated to exhibit substantial in vitro cytotoxicity toward mammalian cells. Furthermore, the requirement for proper disulfide bond formation can reduce the yield of active defensins synthesized since the active molecule contains three intramolecular disulfide bonds.
Thus, there exists a need for an effective microbicidal peptide that can be easily synthesized in an active form and that is effective against a broad spectrum of microorganisms and does not exhibit undesirable side effects. The present invention satisfies these needs and provides related advantages as well.